Stability Of Chloroperoxidase Research Articles

Biocatalytic Performance of Chloroperoxidase from Caldariomyces fumago Immobilized onto TiO2 Based Supports

Chloroperoxidase (CPO) from Caldariomyces fumago is a versatile enzyme able to catalyze the styrene epoxidation. However, the lack of long-term operational stability is the principal drawback for the industrial applications of this enzyme. In this work, in order to increase the enzyme operational stability, CPO immobilization in TiO2 mesoporous materials was studied. Enzyme immobilization onto two TiO2 supports was assayed: microstructured TiO2 anatase (TiO2AN) and nanostructured TiO2 nanotubes (TiO2NT). The CPO/TiO2NT preparation showed 15-times higher catalytic activity for styrene epoxidation than the free enzyme, showing an excellent performance during the styrene epoxidation. In addition, the total turnover numbers for styrene epoxidation, for the free and immobilized enzyme were determined. The CPO immobilization in TiO2 materials increased the operational stability of CPO in 51 % in the case TiO2AN and 69 % for TiO2NT support. These results clearly show that CPO immobilization onto TiO2NT support significantly enhances the CPO enzymatic epoxidation of styrene.

Enhancement of operational stability of chloroperoxidase from Caldariomyces fumago by immobilization onto mesoporous supports and the use of co-solvents

Chloroperoxidase (CPO) from Caldariomyces fumago is a versatile enzyme because of its ability to catalyze different reactions, with high regio- and enantio-selectivities, and among them the styrene epoxidation is of especial industrial interest. However, the use of CPO at industrial scale has been limited mainly due to their low operational stability. In this work, two fundamental aspects are addressed: the selection of a suitable co-solvent to improve biocatalytic epoxidation reaction of hydrophobic substrates such as styrene and some of its derivatives, and CPO immobilization in mesoporous materials in order to increase the enzyme operational stability. Co-solvents tested were acetonitrile, 2-methyl-2-propanol and 2-methyl-2-butanol. In addition, CPO immobilization onto three mesoporous materials was assayed: two siliceous mesocellular foams (MCF and MSU-F) and titanium dioxide (TiO2, anatase). The CPO/MCF preparation showed 3-times higher catalytic activity for styrene epoxidation than the free enzyme. In addition, the total turnover numbers (TTN) were determined for styrene, 3-chlorostyrene and 3-nitrostyrene epoxidations, both with free and immobilized enzyme. The CPO immobilization in mesoporous materials together with use of co-solvent increased the operational stability of CPO in 51% in the case of styrene, 41% for 3-chlorostyrene and 246% for 3-nitrostyrene, when compared to the corresponding values obtained with the free enzyme. These results clearly indicated that CPO immobilization onto nanostructured materials and the use of co-solvent significantly enhances the enzymatic epoxidation reactions of styrene derivatives.

Activity and Stability of Chloroperoxidase in the Presence of Small Quantities of Polysaccharides: A Catalytically Favorable Conformation Was Induced

Chloroperoxidase (CPO) is thought to be the most versatile heme-containing enzyme with enormous applications in organic synthesis, biotransformation, pharmaceutical production, and detoxification of environmental pollutants. Any improvement in the stability of this enzyme will greatly enhance its application in the mentioned areas. In the present study, the effects of three polysaccharides (soluble starch, β-cyclodextrin, and dextrin) on the stability of CPO at elevated temperatures (20, 30, 35, 40, and 50 °C) or in aqueous-organic solvents media (methanol, dioxane, DMSO, and DMF) were investigated. An improved catalytic performance of CPO was observed in the presence of a small amount of the three polysaccharides, where dextrin provided the most effective promotion. The changes of enzyme structure and microenvironment around heme in the presence of additives were studied by fluorescence, circular dichroism, and UV-vis spectra analyses, as well as kinetic parameters measurement. A catalytically favorable structure of CPO was induced, including the strengthening of the α-helix structure and more exposure of heme for easy access of the substrate, resulting in an increase of catalytic turnover frequency (k (cat)) and the improvement of affinity and selectivity of CPO to substrate. The results revealed that the introduction of trace soluble starch, β-cyclodextrin, and dextrin (<10 μmol/L) in reaction media was an effective strategy for the enhancement of the thermodynamic and the operational stability of the enzyme, which are promising in view of the industrial applications of this versatile biological catalyst.

Effects of Additives on the Thermostability of Chloroperoxidase

The effects of several polyhydroxy compounds (glucose, fructose, gumsugar, galactose, trehalose, dextran, xylose, PEG200, glycerin) and surfactant (dioctyl sulfosuccinate sodium salt, AOT) on the catalytic activity and thermal stability of chloroperoxidase (CPO) in aqueous systems were investigated at various temperatures. A 25% superactivity was found in AOT solutions at 25 degrees C, and it could be maintained during the 882 h. PEG200 and glycerin were proven to be the most efficient stabilizer for CPO in temperatures ranging from 25 to 60 degrees C. Trehalose is more helpful than other sugars for extended storage of CPO. These results are promising in view of industrial applications of this versatile biological catalyst. The protective mechanism of various additives on CPO was discussed.

Enhanced catalytic performance and stability of chloroperoxidase from Caldariomyces fumago in surfactant free ternary water–organic solvent systems

Surfactant free ternary systems formulated with organic solvents such as α-pinene or n-hexane, short chain alcohols and water, were used as new reaction media to maximize the catalytic performance of chloroperoxidase from Caldariomyces fumago (CPO) towards the oxidation of hydrophobic compounds including olefins and terpenols. The influence of the composition and the hydration state of the system, the nature of the oxidizing agent as well as substrate availability on the catalytic behavior and stability of the enzyme was investigated, using the chlorination of monochlorodimedone (MCD) and the epoxidation of styrene as model reactions. Based on residual chlorination activity measurements, which were in accordance with differential scanning calorimetry (DSC) findings, it was concluded that in low water systems formulated with α-pinene CPO stability is significantly increased compared to other reaction media (up to 65% residual activity after 24 h of incubation at 30 °C). Electron paramagnetic resonance (EPR) spectroscopy indicated that in low water content systems the ferric environment of the active site of CPO does not undergo severe modifications. Total turnover numbers (TTN) obtained for the oxidation of various olefins and terpenols in α-pinene-based systems, were up to 25,000 which is 2–170 times higher than previously reported values. The results indicate that such ternary systems can be efficiently employed as reaction media leading to highly enhanced stability and increase CPO potential as practical catalyst for oxidation of large amounts of hydrophobic compounds in a single-step biocatalytic process.

Immobilization of chloroperoxidase on mesoporous materials for the oxidation of 4,6-dimethyldibenzothiophene, a recalcitrant organic sulfur compound present in petroleum fractions

The catalytic potential of chloroperoxidase (CPO) immobilized on mesoporous materials was evaluated for the oxidation of 4,6-dimethyldibenzothiophene in water/acetonitrile mixtures. Two different types of materials were used for the immobilization: a metal containing Al-MCM-41 material with a pore size of 26 A and SBA-16 materials with three different pore sizes: 40, 90 and 117 A. The SBA-16 40 A did not retain any CPO. The nature and the pore size of the material affected the catalytic activity of the enzyme as well as its stability. Compared to the free enzyme, the thermal stability of CPO at 45 degrees C was two and three times higher than when immobilized on Al-MCM-41 and SBA-16 90 A, respectively.

Improvement of activity and stability of chloroperoxidase by chemical modification

BackgroundEnzymes show relative instability in solvents or at elevated temperature and lower activity in organic solvent than in water. These limit the industrial applications of enzymes.ResultsIn order to improve the activity and stability of chloroperoxidase, chloroperoxidase was modified by citraconic anhydride, maleic anhydride or phthalic anhydride. The catalytic activities, thermostabilities and organic solvent tolerances of native and modified enzymes were compared. In aqueous buffer, modified chloroperoxidases showed similar Km values and greater catalytic efficiencies kcat/Km for both sulfoxidation and oxidation of phenol compared to native chloroperoxidase. Of these modified chloroperoxidases, citraconic anhydride-modified chloroperoxidase showed the greatest catalytic efficiency in aqueous buffer. These modifications of chloroperoxidase increased their catalytic efficiencies for sulfoxidation by 12%~26% and catalytic efficiencies for phenol oxidation by 7%~53% in aqueous buffer. However, in organic solvent (DMF), modified chloroperoxidases had lower Km values and higher catalytic efficiencies kcat/Km than native chloroperoxidase. These modifications also improved their thermostabilities by 1~2-fold and solvent tolerances of DMF. CD studies show that these modifications did not change the secondary structure of chloroperoxidase. Fluorescence spectra proved that these modifications changed the environment of tryptophan.ConclusionChemical modification of epsilon-amino groups of lysine residues of chloroperoxidase using citraconic anhydride, maleic anhydride or phthalic anhydride is a simple and powerful method to enhance catalytic properties of enzyme. The improvements of the activity and stability of chloroperoxidase are related to side chain reorientations of aromatics upon both modifications.

Stabilization of interface-binding chloroperoxidase for interfacial biotransformation

The stability of an interface-binding chloroperoxidase (CPO) against the deactivation effect of H(2)O(2) was examined. Native CPO was conjugated with polystyrene and thus self-assembled at the water-oil interface. Although the interface-assembled CPO showed improved stability as compared to native CPO, enzyme deactivation as a result of the side effect of H(2)O(2), still limits the overall productivity of the enzyme. Two approaches to further improve the stability of CPO were examined in this work. In one approach, several stabilizers including poly(ethylene glycol) (PEG), PEI, glycerol, sugars and sucrose monododecanoate were used; while in a second approach, in situ generation of hydrogen peroxide (H(2)O(2)) by using glucose oxidase (GOx) was applied. PEG was found exceptional in that it increased both the operational and storage stability of CPO. The best improvement of enzyme productivity was obtained with addition of PEG which led to an increase of 57% for interface-bound CPO and 33% for native CPO. One interesting observation with PEI is that it enhanced the storage stability against H(2)O(2) deactivation, but did not affect the enzyme's operational stability. On the other hand, glucose enhanced the operational stability by two folds, but exhibited no significant effect on storage stability. It was also found that the extended operational lifetime of CPO with in situ generation of H(2)O(2) by GOx was a result that combines the stabilizing effect of glucose and lowered concentration of H(2)O(2). Interestingly, the addition of stabilizers could improve the enantioselectivity of CPO by as much as 10%.